S. Sabattini

An immunohistochemical analysis of canine haemangioma and haemangiosarcoma.

The aim of the present study was to investigate immunohistochemically aspects of the biology of canine endothelial neoplasia. Forty samples of canine cutaneous and visceral haemangiosarcoma (HSA), 29 samples of cutaneous and visceral haemangioma (HA) and 10 control samples of granulation tissue (GT) were labelled with antisera specific for vimentin, smooth muscle actin, von Willebrand factor (vWF), CD117 (KIT), vascular endothelial growth factor receptor-3 (VEGFR-3), vascular endothelial growth factor-C (VEGFC) and CD44. Further antisera were employed to determine the level of cellular proliferation (MIB-1 index) and toluidine blue staining was used to detect populations of tumour-infiltrating mast cells (MCs). There was greater expression of CD117, VEGFR-3 and CD44 in HSA than in HA, suggesting that these proteins might be suitable targets for the future development of novel therapeutic approaches to canine HSA. Marked infiltration of MC was detected in HA, suggesting a possible role for these cells in the pathogenesis of benign vascular neoplasia in the dog.

Histologic Grading of Canine Mast Cell Tumor Is 2 Better Than 3

Mast cell tumor (MCT) is a common canine cutaneous neoplasm with variable biological behavior. A 2-tier histologic grading system was recently proposed by Kiupel et al to reduce interobserver variation and eliminate prognostic uncertainty of the Patnaik system. This study compared the ability of these 2 grading systems to predict survival in a cohort of dogs with MCTs. However, surgical margins were unknown, and the risk of developing new/metastatic MCTs was not studied. Histologic grade was assessed according to both systems for 137 surgically resected cutaneous MCTs. The relationship between grade and survival was evaluated. According to the Patnaik system, 18 MCTs (13.1%) were classified as grade I, 83 (60.6%) as grade II, and 36 (26.3%) as grade III. Grade III was associated with a poorer prognosis (P < .001), but no significant difference between grades I and II was detected. Grading according to the Patnaik system was based on consensus grading among 3 pathologists, and interobserver variability was not considered. All grade I MCTs were low grade in the Kiupel system, and all grade III were high grade. Among grade II, 71 (85.6%) were low grade, and 12 (14.4%) were high grade, with a 1-year survival probability of 94% and 46%, respectively (P < .001). The 2-tier system had a high prognostic value and was able to correctly predict the negative outcomes of some grade II MCTs. Data also confirm that histologic grading cannot predict biological behavior of each MCT and should be supplemented with molecular methods for more accurate prognostication.

Prognostic Value of Histologic and Immunohistochemical Features in Feline Cutaneous Mast Cell Tumors

Feline cutaneous mast cell tumors (MCTs) have been histologically classified as mastocytic (well differentiated or pleomorphic) and atypical/poorly granulated. Their biologic behavior ranges from benign to malignant, but prognostic factors are not well defined. Histologic classification, number of tumors, mitotic index, cytoplasmic granularity, and infiltration by eosinophils or lymphocytes were evaluated retrospectively in 25 feline cutaneous MCTs. Immunohistochemistry was applied to assess KIT (CD117) pattern and immunoreactivity score, telomerase expression (human telomerase reverse transcriptase), and proliferation index (MIB-1/Ki67 index). Case outcome was obtained via telephone interviews. The tumors comprised 15 mastocytic well-differentiated, 7 mastocytic pleomorphic, and 3 atypical/poorly granulated MCTs. Immunohistochemically, CD117 was expressed in 13 of 25 tumors (52%), and telomerase reverse transcriptase was expressed in 15 of 22 (68%), with no correlation to histologic classification. Mitotic index, KIT immunoreactivity score, and Ki67 index were significantly higher in mastocytic pleomorphic MCTs than in the other 2 categories. Five cats (20%) died of tumor-related causes. Multiplicity of lesions, pleomorphic phenotype, KIT immunoreactivity score, and mitotic and Ki67-indices correlated with an unfavorable outcome. Mitotic index was the strongest predictive variable. These results suggest that histologic classification, CD117/KIT immunohistochemistry, and proliferation indices may help to identify potentially aggressive cases of feline cutaneous MCT. Aberrant KIT protein localization and telomerase immunoreactivity warrant further exploration as potential prognostic markers.

Use of histologic margin evaluation to predict recurrence of cutaneous malignant tumors in dogs and cats after surgical excision.

OBJECTIVE To assess the usefulness of histologic evaluation of surgical margins to predict local recurrence of cutaneous malignant tumors in dogs and cats treated by means of surgical excision. DESIGN Prospective case series. ANIMALS 40 dogs and 20 cats. PROCEDURES 60 surgically excised tumors (20 soft tissue sarcomas [STSs], 20 mast cell tumors [MCTs], and 20 carcinomas) were examined histologically. Margins were classified as clean, close, or infiltrated; histologic grade was assessed in STSs and MCTs. Recurrence rates and recurrence-free intervals (RFIs) during a 24-month follow-up period were recorded, and method accuracy was calculated. RESULTS Surgical margins were clean in 29 of 60 (48%) tumors, close in 11 (18%), and infiltrated in 20 (33%). Tumors recurred in 27 of 60 (45%) animals, with a mean ± SD RFI of 229 ± 173 days. Recurrence rates for animals that had tumors with infiltrated (16/20) or close (8/11) margins were significantly higher than recurrence rate for animals that had tumors with clean margins (3/29). Margin classification was a significant predictor of RFI. Accuracy of the method to predict recurrence was 94% for carcinomas, 87% for STSs, and 76% for MCTs. CONCLUSIONS AND CLINICAL RELEVANCE Histologic assessment of margin status was useful for predicting local recurrence of cutaneous malignant tumors in dogs and cats treated by means of excision alone. Method accuracy varied among tumor types and grades. Recurrence times suggested postsurgical follow-up should continue for ≥ 2 years. Results were similar for animals with infiltrated and close tumor margins, and careful postsurgical management is recommended for both.

Comparison of 2- and 3-category histologic grading systems for predicting the presence of metastasis at the time of initial evaluation in dogs with cutaneous mast cell tumors: 386 cases (2009–2014)

OBJECTIVE To compare the Kiupel (2 categories) and Patnaik (3 categories) histologic grading systems for predicting the presence of metastasis at the time of initial examination in dogs with cutaneous mast cell tumors (MCTs). DESIGN Retrospective case series. ANIMALS 386 client-owned dogs with cutaneous MCTs. PROCEDURES Medical records of dogs with newly diagnosed, histologically confirmed cutaneous MCTs that had undergone complete clinical staging were reviewed for clinical and histopathologic data. RESULTS All Patnaik grade 1 MCTs (n = 52) were classified as Kiupel low-grade MCTs, and all Patnaik grade 3 MCTs (43) were classified as Kiupel high-grade MCTs. Of the 291 Patnaik grade 2 MCTs, 243 (83.5%) were classified as Kiupel low-grade tumors, and 48 (16.5%) were classified as Kiupel high-grade MCTs. Dogs with Patnaik grade 3 MCTs were significantly more likely to have metastases at the time of initial examination than were dogs with grade 1 or 2 MCTs (OR, 5.46), and dogs with Kiupel high-grade MCTs were significantly more likely to have metastases than were dogs with Kiupel low-grade MCTs (OR, 2.54). However, 3 of 52 (5.8%) dogs with Patnaik grade 1 tumors, 48 of 291 (16.5%) dogs with Patnaik grade 2 tumors, and 44 of 295 (14.9%) dogs with Kiupel low-grade tumors had metastatic disease. CONCLUSIONS AND CLINICAL RELEVANCE Findings indicated that in dogs with cutaneous MCTs, prognostication should not rely on histologic grade alone, regardless of grading system used, but should take into account results of clinical staging.

Epidermal growth factor receptor expression is predictive of poor prognosis in feline cutaneous squamous cell carcinoma

The aim of this study was to investigate epidermal growth factor receptor (EGFR) expression in feline cutaneous squamous cell carcinoma (FC-SCC) and assess its prognostic role. Nineteen formalin-fixed paraffin-embedded excisional biopsies of FC-SCC were tested for EGFR expression using immunohistochemistry (IHC). Relationships between EGFR expression and histopathological parameters (differentiation, mitotic activity), disease-free interval (DFI) and overall survival (OS) at 24 months were further investigated. Fourteen of 19 tumours (73.7%) were positive for EGFR, with great variation in intensity and proportion of labelled cells. EGFR expression was not correlated with tumour differentiation or mitotic activity. Nine cats (47.4%) died of tumour-related causes. Patients with EGFR-positive tumours had a significantly worse outcome (P=0.0217), with decreased DFIs (P=0.0075) and survival times (P=0.0391). These data suggest that EGFR expression carries a negative prognostic significance in FC-SCC. EGFR inhibitors in association with conventional treatments may improve outcome for the subgroup of cats with EGFR-positive tumours.

EGFR, HER-2 and KRAS in Canine Gastric Epithelial Tumors: A Potential Human Model?

Epidermal growth factor receptor (EGFR or HER-1) and its analog c-erbB-2 (HER-2) are protein tyrosine kinases correlated with prognosis and response to therapy in a variety of human cancers. KRAS mediates the transduction of signals between EGFR and the nucleus, and its mutation has been identified as a predictor of resistance to anti-EGFR drugs. In human oncology, the importance of the EGFR/HER-2/KRAS signalling pathway in gastric cancer is well established, and HER-2 testing is required before initiating therapy. Conversely, this pathway has never been investigated in canine gastric tumours. A total of 19 canine gastric epithelial neoplasms (5 adenomas and 14 carcinomas) were retrospectively evaluated for EGFR/HER-2 immunohistochemical expression and KRAS mutational status. Five (35.7%) carcinomas were classified as intestinal-type and 9 (64.3%) as diffuse-type. EGFR was overexpressed (≥1+) in 8 (42.1%) cases and HER-2 (3+) in 11 (57.9%) cases, regardless of tumour location or biological behaviour. The percentage of EGFR-positive tumours was significantly higher in the intestinal-type (80%) than in the diffuse-type (11.1%, p = 0.023). KRAS gene was wild type in 18 cases, whereas one mucinous carcinoma harboured a point mutation at codon 12 (G12R). EGFR and HER-2 may be promising prognostic and therapeutic targets in canine gastric epithelial neoplasms. The potential presence of KRAS mutation should be taken into account as a possible mechanism of drug resistance. Further studies are necessary to evaluate the role of dog as a model for human gastric cancer.

Prognostic significance of Kit receptor tyrosine kinase dysregulations in feline cutaneous mast cell tumors.

Feline cutaneous mast cell tumors (FeCMCTs) are characterized by variable biological behavior. Development of multiple nodules and potential visceral involvement, along with inconsistency of conventional prognostic aids, justify uncertainty in differentiating benign from malignant forms. c-Kit proto-oncogene activating mutations have been reported in feline mast cell tumors (MCTs), but their prognostic relevance was not investigated. This study was performed on FeCMCTs with variable clinical outcome to assess whether Kit cytoplasmic immunohistochemical labeling can be regarded as indicative of c-Kit mutations and to evaluate the relationship between Kit dysregulation and survival. Twenty-four cats diagnosed with a primary cutaneous MCT were enrolled. Kit immunohistochemical pattern and c-Kit (exons 8, 9, 11) mutational status were assessed in 34 tumor samples. Risk factors affecting survival were a number of mitoses greater than 5 per 10 HPFs (P = .017) and cytoplasmic Kit labeling (P = .045). Increased mitotic activity was associated with Kit cytoplasmic expression (P = .01). c-Kit encoding mutations were present in 19 (56%) tumors (exon 8, 19%; exon 9, 71%; exon 11, 10%), however, they were not significantly related to protein expression and they had no influence on prognosis. Additionally, in 6 of 9 (67%) cats, multiple nodules from the same cat had different mutational statuses. Mutations in the fifth immunoglobulin-like domain of Kit occur frequently in FeCMCT, but they are variably associated with aberrant protein expression and do not appear to be strictly correlated with biological behavior. These findings need to be confirmed in larger series, and exploration of further genomic regions of c-Kit is warranted.

Cytological grading of canine cutaneous mast cell tumours.

A cytological grading for mast cell tumours (MCTs) would be highly desirable, allowing to select the most appropriate therapeutic intervention prior to surgery. This study evaluates the applicability on fine-needle aspirations (FNAs) of the novel Kiupel grading system, based on number of mitoses, multinucleated cells, bizarre nuclei and presence of karyomegaly. Fifty consecutive cases with pre-operative cytological diagnosis were included. In cytological specimens, approximately 1000 cells were evaluated, and the histological grade was assessed on the corresponding resected specimens. On cytology, the above parameters were significantly different between histologically low-grade and high-grade tumours (P < 0.001). The cytograding correctly predicted the histological grade in 47 cases (accuracy, 94%; sensitivity, 84.6%; specificity, 97.3%). Two high-grade MCTs (4%) were not detected on cytology. The cytograding can provide helpful insights to assist clinical decisions in most cases. However, the risk of underestimation in a minority of patients represents a limit to the overall utility of the technique.

EGFR overexpression in canine primary lung cancer: pathogenetic implications and impact on survival†

This study reports the main clinicopathological features of primary lung cancer (PLC) in 37 dogs, with special regard to the pathogenetic and prognostic role of epidermal growth factor receptor (EGFR) overexpression. For each case the following characteristics were evaluated: tumour-node-metastasis (TNM) stage, tumour histotype, histological grade, mitotic activity and immunohistochemical expression of EGFR. In samples with available normal lung tissue, the amount of background anthracosis was also measured by image analysis. In 27 tumours (73%) a variable number of cells (20-100%) stained positively for EGFR. The proportion of EGFR-positive tumours was significantly higher in cases with background anthracosis, and the amount of anthracosis was correlated with the percentage of positive tumour cells. Additionally, a trend towards shortened survival for the high EGFR group was observed. These findings suggest an involvement of EGFR signalling pathway in canine PLC, a negative prognostic significance of protein overexpression and its potential implication in air pollution carcinogenesis.